Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly form of liver cancer. Beyond surgical resection which can only be applied to a minority of the patients, there is no effective treatment for ICC. There is a clear urgent need to develop novel treatment strategies against this deadly malignancy. Recently, we and others demonstrated the critical role of Notch and Hippo signaling cascades in ICC pathogenesis. For the Notch pathway, we found that Notch receptors, including Notch1 and Notch2 are highly expressed in majorities of human ICC samples. Treatment of ?-secreatase inhibitors (GSI) or inhibition of Notch using molecular approaches is able to repress human ICC cell proliferation and induce apoptosis in vitro. Mechanistically, we showed that Notch functions to regulate MAPK signaling. For the Hippo pathway, we uncovered a strong and ubiquitous activation of Yap and TAZ, the transcriptional activators downstream of Hippo tumor suppressor kinases, in human ICC as well as mouse ICC samples. In vitro, upregulation of Hippo cascade by overexpressing Lats2 or blocking Yap/TAZ activity via dominant negative form of TEAD2 (dnTEAD2) strongly inhibited ICC cell growth. Similar results were obtained when Yap or TAZ expression was silenced in human ICC cells. Furthermore, overexpression of Lats2 or dnTEAD2 strongly delayed ICC development induced by loss of Pten and TP53 tumor suppressors (sgPten/sgP53). In vivo, using hydrodynamic transfection, we discovered that activated forms of Yap (YapS127A) or TAZ (TAZS89A) synergized with activated AKT signaling to promote ICC development. ICC driven by activated AKT and Yap/TAZ depends on Notch as blocking canonical Notch strongly inhibited ICC development in mice. Based on these preliminary studies, we hypothesize that the canonical Notch pathway medicated via Notch1 and/or Notch2 is required for ICC development via modulating MAPK cascade. We further hypothesize that Yap and TAZ, the major transcriptional activators downstream of Hippo tumor suppressor kinases, have overlapping yet distinct roles in ICC development and elucidation of these roles will be critical to exploit the modulation of this pathway for translational implications. Two aims are proposed to test these hypotheses. In Aim 1, we will elucidate the canonical Notch signaling cascade in ICC development. In Aim 2, we will define the functional contribution of Yap and TAZ in regulating ICC pathogenesis. Altogether, in the proposed application, we will apply in vitro mechanistic studies in combination with sophisticated mouse genetic approaches to uncover the molecular mechanisms underlying Notch and Hippo cascades in regulating ICC tumorigenesis. The study will likely provide strong evidence to support the development and application of anti-Notch and/or Yap/TAZ based therapeutics for ICC treatment. Based on the quality of preliminary data and availability of all necessary reagents for balanced in vitro and in vivo studies, we feel confident that we will be able to address the significance of these biological pathways in liver pathology to eventually devise better understanding and hence therapies for ICC.